Difference between revisions of "User:Wesley J. Houston/Notebook/MAP/2013/02/08"

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(February 8, 2013)
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Line 12: Line 12:
 
----
 
----
 
'''Assemblies'''
 
'''Assemblies'''
# KAH201/KAH182: (1) KAH201/(E/S)/203 + (2) KAH182/(E/X)/1675
+
# KAH201/KAH182: (1) KAH201/(S/P)/203 + (2) KAH182/(X/P)/1675
  
 
> Digests (Fermentas FD)<br>
 
> Digests (Fermentas FD)<br>
Line 20: Line 20:
 
|-valign="top"
 
|-valign="top"
 
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
 
| <u>Reagent</u> || <u>Volume</u> || &nbsp;
| rowspan="9" | [[Image:KAH113012_gel1.jpg‎ |150px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
+
| rowspan="9" | [[Image:KAH113012_gel1.jpg‎ |150px|Placeholder Image]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
 
|-
 
|-
| DNA (plasmid) || 15 to 25 μL
+
| DNA (plasmid) || 20 μL
 
|-
 
|-
 
| 10x buffer || 3.0
 
| 10x buffer || 3.0
Line 30: Line 30:
 
| enzyme 2 || 1.0
 
| enzyme 2 || 1.0
 
|-
 
|-
| dH<sub>2</sub>O || 0 to 10 μL
+
| dH<sub>2</sub>O || 5.0 μL
 
|-
 
|-
 
| &nbsp; || 30 μL --> 37°C/ ~15 min.
 
| &nbsp; || 30 μL --> 37°C/ ~15 min.
Line 41: Line 41:
 
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
 
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
 
|-
 
|-
| 1. KAH201 (E/S) || TBD || TBD || TBD
+
| 1. KAH201 (S/P) || 0.048 || 1.88 || 48.1
 
|-
 
|-
| 2. KAH182 (E/X) || TBD || TBD || TBD
+
| 2. KAH182 (X/P) || 0.008 || 1.93 || 8.1
 
|}
 
|}
  
  
> Ligations (edit after ligation is done)
+
> Ligations
 
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
 
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
 
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
 
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
 
|-
 
|-
| 1. KAH201 (E/S)/203, x ng + KAH182 (E/X)/1675, x ng || <font color="blue">---</font>
+
| 1. KAH201 (S/P)/203, 25 ng + KAH182 (X/P)/1675, 6 ng || <font color="blue">TBD</font>
 
|-
 
|-
| 2. KAH182 (E/X)/ x ng || ---
+
| 2. KAH182 (X/P)/1675 6 ng || TBD
 
|}
 
|}
  
Line 59: Line 59:
 
| &nbsp;            || 1    || 2    ||
 
| &nbsp;            || 1    || 2    ||
 
|-
 
|-
| Insert DNA        || --- || ---  ||
+
| Insert DNA        || 0.74 || ---  ||
 
|-
 
|-
| Vector DNA        || --- || --- ||
+
| Vector DNA        || 0.52 || 0.52 ||
 
|-
 
|-
| 2x lgn buf (Roche) || --- || --- ||
+
| 2x lgn buf (Roche) || 5.0 || 5.0 ||
 
|-
 
|-
| T4 ligase (NEB)    || --- || --- ||
+
| T4 ligase (NEB)    || 1.0 || 1.0 ||
 
|-
 
|-
| dH<sub>2</sub>O    || ---  || --- ||
+
| dH<sub>2</sub>O    || 2.74 ||  3.48 ||
 
|-
 
|-
 
| &nbsp;            || 10 μL || 10 μL ||
 
| &nbsp;            || 10 μL || 10 μL ||

Revision as of 13:59, 21 February 2013

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February 8, 2013

  • Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins.



Assemblies

  1. KAH201/KAH182: (1) KAH201/(S/P)/203 + (2) KAH182/(X/P)/1675

> Digests (Fermentas FD)


Reagent Volume   Placeholder Image
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 5.0 μL
  30 μL --> 37°C/ ~15 min.


> Measure conc.'s of gel purified fragments

Sample OD260 260/280 ng/μL
1. KAH201 (S/P) 0.048 1.88 48.1
2. KAH182 (X/P) 0.008 1.93 8.1


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH201 (S/P)/203, 25 ng + KAH182 (X/P)/1675, 6 ng TBD
2. KAH182 (X/P)/1675 6 ng TBD
  1 2
Insert DNA 0.74 ---
Vector DNA 0.52 0.52
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.74 3.48
  10 μL 10 μL
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar